Boraginaceae) Endemic to Sardinia (Italy

Systematics and Biodiversity 6 (2): 161–174 Issued 6 June 2008 doi:10.1017/S1477200008002673 Printed in the United Kingdom C The Natural History Museum

G. Bacchetta1 , A. Coppi2 , ∗ C. Pontecorvo1 &F.Selvi2 Systematics, phylogenetic relationships 1Centro Conservazione Biodiversità (CCB), and conservation of the taxa of Anchusa Dipartimento Scienze Botaniche, University of (Boraginaceae) endemic to Sardinia (Italy) Cagliari, Viale Sant’Ignazio da Laconi, 13 – I09123, Cagliari, Italy Emails: [email protected]; [email protected] Abstract Sardinia is a major centre of diversity of Anchusa, but the systematics, 2Dipartimento di Biologia phylogenetic relationships and conservation status of the taxa endemic to the island Vegetale, Sezione Botanica Sistematica, University of are still poorly known mainly because of their remarkable rarity. We present a Firenze, Via La Pira, 4 – I 50121, study on these endemics based on the results of field research focusing on the Firenze, Italy distribution, the number and size of the populations, the characteristics of the habitat submitted January 2006 and the factors of threat. Combined with observations on aspects of reproductive accepted September 2006 biology, on wild populations and cultivated plants, we evaluate the conservation status of the taxa and propose Red List IUCN categories of vulnerability. Original descriptions, nomenclatural types and karyological data are added. The following Anchusa taxa are endemic to Sardinia: A. capellii, A. crispa ssp. crispa, A. crispa ssp. maritima, A. formosa, A. littorea, A. sardoa and A. montelinasana sp. nov. The latter is described, based on the discovery of a morphologically distinct entity on a mountain in southwest Sardinia. Anchusa littorea was found at a single site after c. 25 years from the last record and 7 years of unsuccessful field research; this species is at the brink of extinction due to the extremely reduced size of the only remaining population. Our complete collection of taxa allowed an analysis of phylogenetic relationships based on DNA sequences from the ITS1 ribosomal genome. In spite of the low variation, this marker produced Maximum Parsimony and Neighbour-Joining phylograms suggesting that the group is monophyletic and that the split between the two clades of the mountain and coastal endemics has been a key evolutionary event. We assume the three mountain species to be relict schizoendemics ancestral to the coastal taxa, and the Paleozoic siliceous massifs of central and south Sardinia as the centre of origin of the group.

Key words Anchusa, Boraginaceae, conservation, endemism, molecular phylogeny, Sardinia, systematics

Introduction lation size, which explains the lack of herbarium material in many European collections and, consequently, the uncertain Anchusa L. is one of the major genera of tribe Boragineae and often discordant taxonomic interpretations in past and re- (Boraginaceae), with approximately 30 species mainly dis- cent standard floras (Fiori, 1926; Chater, 1972; Pignatti, 1982; tributed in the Mediterranean basin and Middle East, and Greuter et al., 1984). Perhaps the only exception is represented three disjunct African members in the highlands of Eritrea by the psammophytic species, A. crispa Viv., which is the only and Cape region (Gus¸uleac, 1929). Sardinia is a major centre member of the group also occurring on the nearby island of of diversity and endemism for this genus, with six allopatric Corsica. The narrow range, the small size of the populations, taxa occurring in either coastal or mountain habitats of the the loss of habitat caused by human activities and the negative island. However, despite some previous taxonomic contribu- effects of other biotic and abiotic factors are the main causes for tions (Valsecchi, 1976; Selvi, 1998; Selvi & Bigazzi, 1998), which this species is listed in the French and Italian National these narrow-ranged endemics are still poorly known in terms Red Lists (Olivier et al., 1995; Conti et al., 1997; Scoppola & of both phylogenetic relationships and conservation status. A Spampinato, 2005), in the EU Directive 92/43 CEE ‘Habitat’, reason for this is their very restricted range and small popu- in the Berne Convention and finally in a recent IUCN selec- tion of the 50 most endangered species of the Mediterranean ∗Corresponding author. Email: selvi@unifi.it (De Montmollin & Strahm, 2005). Accordingly, A. crispa has

161 162 G. Bacchetta et al. been the object of numerous studies on different ecological number of sites recorded previously for each taxon on the and reproductive aspects related to the problems of its in situ basis of reliable literature data and herbarium specimens in FI, conservation, though almost exclusively on Corsica (Thiebaut,` CAG, SASSA, TO and G. For the rarest species we prepared 1988; Guyot & Muracciole, 1995; Paradis & Piazza, 1988, fine scale maps, which will allow the detection of eventual 2000; Quilichini & Debussche, 2000; Quilichini et al., 2001, future changes in the area of occupancy of the populations. 2004). Based on these data, we propose conservation categories for It is evident, therefore, that the disparity of knowledge each taxon following the IUCN criteria (IUCN, 2001). In ad- that we have for this ‘target’ species with respect to the other dition, collection of seed allowed us to make preliminary tests Sardinian endemics even in basic terms of number and surface on germination capacity and to grow plants in the botanical of the populations is not a favourable condition to plan initiatit- garden for the purposes of ex situ conservation. During the ives for their long-term, in-situ conservation. To fill this gap we years 2000–2005 we monitored the production of viable seed undertook a field-based research to obtain more information in isolated plants under cultivation. on the distribution, habitat features and demographic condi- Before treating the above-mentioned aspects, we give for tion of the populations. Seven years of field work allowed us each taxon: (1) the nomenclatural type; (2) a brief morpholo- to find new localities for some of the rarest taxa, to better gical description based on field notes and herbarium material; understand the biotic and/or abiotic factors that influence the (3) references to selected iconographies and an original illus- demographic dynamics of populations, and to establish cor- tration of the new species A. montelinasana. The latter species rect IUCN categories. This is the essential information to plan and the new population of A. formosa from Mount Arcosu recovery actions of all endangered species (Cropper, 1993; (see below) were also analysed from a karyological viewpoint, Frankel et al., 1995). In addition, we discovered a popula- using root tips of seeds germinated in Petri dishes. These were tion of Anchusa in a rugged mountain area of the southwest- pre-treated with 0.002 M 8-hydroxyquinoline or 0.05% col- ern part of the island, whose morphological distinctiveness chicine, 2.5 h at room temperature and then fixed overnight allowed us to describe it as a new species. Such an import- in ethanol:glacial acetic acid 3:1. The meristematic tissue was ant discovery made possible a complete collection of mater- then thoroughly rinsed in distilled water, hydrolysed in 1N ial for the phylogenetic analysis of the group using sequences HCl at 60 ◦C for 6–7 minutes, and stained in lacto-propionic from the non-coding Internal Transcribed Spacer region (ITS1) orcein overnight. Meristems were finally dissected, squashed of the nuclear ribosomal DNA. The usefulness of this molecu- on clean glass slides in a drop of 45% acetic acid, and observed lar marker in the species-level systematics of Boragineae has with a Zeiss Axioscop light microscopy under oil immersion been demonstrated in recent studies (Hilger et al., 2004; Selvi (100×). et al., 2004). The results of this work are reported in this paper in order to contribute to a better knowledge and to the con- DNA isolation and amplification servation of this remarkable component of the Mediterranean Genomic DNA of the taxa listed in Table 1 was extracted × insular endemic flora. following a modified 2 CTAB protocol (Doyle & Doyle, 1990) using silica-gel samples of leaf tissue collected in the field by the authors. Voucher specimens are deposited in FI, Materials and methods CAG and BM. The extracted DNA was quantified after agarose gel electrophoresis (0.6% w/v) in TAE buffer (1 mM EDTA, Fieldwork and plant material 40 mM Tris-acetate) containing 1 μg/ml of ethidium bromide This account is mainly based on fieldwork (1998–2005) fo- by comparison with a known mass standard. The primers ITS4 cusing on the distribution, ecology and conservation status of and ITS5 (Baldwin, 1992) were used for the amplification of the taxa in terms of number and area of occupancy of the the Internal Transcribed Spacer ITS1 region of nuclear rDNA populations, approximate number of fertile individuals, hab- intron. PCR amplifications were performed in a total volume itat characteristics and main factors of threat. When possible, of 50 μl containing 5 μl of reaction buffer (Dynazyme II; we captured the insects that visited the flowers and made ob- Finnzyme, Espoo, Finland), 1.5 mM MgCl2, 20 pmol of each servations on the means of seed dispersal. We determined the primer, 200 μM of each dNTP, 1 U of Taq DNA polymerase

Taxa Geographic origin and herbarium vouchers A. capellii Moris Sardinia, Esterzili, Monte S. Vittoria, G. Bacchetta & F. Selvi, 99.002 (FI) A. crispa Viv. ssp. crispa Corsica, Solenzara, Cannella, M. Bigazzi & F. Selvi, 99.005 (FI) A. crispa Viv. ssp. crispa Sardinia, Alghero, Porticciolo, M. Bigazzi & F. Selvi, 97.001 (FI) ““ ssp. maritima (Vals.) Selvi & Bigazzi Sardinia, Badesi, mouth of river Coghinas, M. Bigazzi & F. Selvi, 97.005 (FI) A. formosa Selvi, Bigazzi & Bacchetta Sardinia, Uta, Mt. Lattias, M. Bigazzi & F. Selvi, 97.006 (FI, CAG) A. littorea Moris Sardinia, Arbus, Is Arenas, G. Bacchetta & C. Pontecorvo (CAG) A. montelinasana Sardinia, Gonnosfanadiga, Mt. Linas, C. Pontecorvo & R. Angius (CAG, FI) A. sardoa (Illario) Selvi & Bigazzi Sardinia, Alghero, Porto Conte, M. Bigazzi & F. Selvi, 97.020 (FI)

Table 1 List of the taxa of Anchusa and accessions used for the molecular analysis. Systematics, phylogenetic relationships and conservation of the taxa of Anchusa (Boraginaceae) endemic to Sardinia (Italy) 163

(Dynazyme II; Finnzyme) and 10 ng of template DNA. Re- actions were performed in a Perkin-Elmer 9600 thermocycler (Perkin Elmer, Norwalk, CT, USA). Then, 5 μl of each amplification mixture was analysed by agarose gel (1.5% w/v) electrophoresis in TAE buffer. The PCR reactions were purified from excess salts and primer with the PCR Purification Kit (Roche, Mannheim, Germany). Automated DNA sequen- cing was performed on both strands directly from the ITS4 and ITS5 primers on the purified PCR products using BigDye Ter- minator v.2 chemistry and an ABI310 sequencer (PE-Applied Biosystems, Norwalk, CT, USA) according to the manufac- turer’s recommendations. Sequence alignment and phylogenetic analysis The nucleotide sequences obtained were checked for ortho- logy to the sequences of Anchusella cretica (Mill.) Bigazzi, Nardi & Selvi (GenBank accession AY045716), Anchusa stylosa M.Bieb. of Anchusa subgenus Buglossellum Gus¸ul. (AY383308) and Anchusa azurea Mill. of Anchusa subg. Bu- glossum Gus¸ul. (AY383293). Based on their position in a recent phylogeny of tribe Boragineae (Hilger et al., 2004), we then used these species as outgroup representatives for dendrogram construction. Multiple alignment was performed with the program Multalin (Corpet, 1988), and then further examined and slightly modified manually. All characters were weighted equally, and character state transitions were treated as unordered. Gaps were coded and added at the end of the sequences according to the ‘simple gap’ coding method after Simmons & Ochoterena (2000). Neighbour-Joining (NJ), and Maximum Parsimony (MP) methods were used to analyse the aligned sequences. NJ trees (Saitou & Nei, 1987) were obtained on the basis of a Kimura-2 parameter distance matrix. Figure 1 Distribution of the endemic Anchusa in Sardinia. Solid MP trees were calculated with PAUP∗ ver. 4.0b (Swofford, symbols refer to confirmed sites; empty symbols refer to historical (not confirmed) sites. 1998) through heuristic search adding sequences at random with Tree-Bisection-Reconnection branch swapping. Accel- erated transformation (ACCTRAN) optimisation was used to infer branch lengths. Internal support to the branches was es- a reticulation of blunt ridges. Flowering during April–June, timated by means of 50% Majority-Rule bootstrap analysis secondarily to July; fruiting during May–July. with 1000 replicates (Felsenstein, 1985). Iconography.SELVI et al. (1997: Fig. 1).

Results Reproductive biology Allogamous pollination is by means of insects. The follow- Anchusa formosa Selvi, Bigazzi & Bacchetta, Pl. Biosys. 131: ing taxa were observed to visit the flowers with high fre- 104. 1997. — Type: holo-FI; iso- FI, CAG, SASSA. quency: Apis mellifera, Halictus sp. (Hymenoptera), Lasiom- Perennial. Hispid for stinging, patent setae up to 3.1 mm long mata tigelius, Coenoympha corinna (Lepidoptera), Bombylius and inserted on prominent tubercles, and sparse, shorter hairs. sp. (Diptera), Divales cinctus, Oedemera sp. (Coleoptera). In Stems up to 40 cm, prostrate-ascending to decumbent. Basal cultivated plants, however, self-pollination plays an important leaves in a dense rosette, 8–18(30) × 2.5–5(8) cm, bright green, role and production of viable seed is abundant. Seed dispersal ovate to oblanceolate, tapering into a short petiole, with entire, is operated by ants and running water along mountain chan- flat margins; cauline leaves smaller, sessile. Cymes branched nels. Seed set is high in either wild or cultivated plants, and distally, scarcely elongated in fruit. Bracts triangular-ovate, percentage of germination is high (c.90%). slightly shorter than calyx. Flowers subsessile, with calyx 5–6 Chorology. SW Sardinian endemic (region of eastern Sulcis) 1 mm long, divided to c. /4. Fruiting calyx ventricose-urceolate, Since its description the species was known for only the Senna costate, with reflexed teeth. Corolla with tube c.5mmand Manna and Su Fundu channels which run down from the east- limb subrotate, c. 8 mm in diameter, light blue-violet. Anthers ern side of Monte Lattias. Our field researches led to the dis- 1.7 mm, partially overlapping scales. Homostylous; style c.5 covery of a second, small population at Su Scavoni, a locality mm. Mericarpids obliquely ovoid, small, c.2× 1.5 mm, with on the north-eastern side of Monte Arcosu c. 4.5 km at the a weak basal rim; surface blackish, minutely papillose, with north from the first one (Figs 1, 2). 164 G. Bacchetta et al.

Figure 2 Area and location of the populations of (top left) A. montelinasana (Mt. Linas); (top right) A. formosa (Mt. Arcosu); (lower left) A. littorea (Is Arenas); (lower right) A. capellii (Mt. Santa Vittoria). A map of the Monte Lattias population of A. formosa is published in Selvi et al. (1997).

Habitat and ecology VU category (Table 3). However, it is not severely threatened The species grows on incoherent material of erosion in rocky with extinction mainly because both populations lie in the channels with an inclination between 5◦ and 45◦. Rock type is heart of a wilderness area which is part of the WWF Nature granite of Hercinian origin. The Mt. Lattias population consists Reserve and site of Comunitarian Interest ‘Monte Arcosu’ of three subpopulations located at c. 550–580 m, 620–680 and (code ITB 041105), and of the regional park ‘Sulcis’ (regional 700–795 m respectively; the Mt. Arcosu site is at 860 m a.s.l. law 31/89). The remarkable demographic fluctuations in the Both are faced to the east and lie in the mesomediterranean up- Mt. Lattias population were caused by natural events. During per subhumid-lower humid bioclimatic belt (upper subhumid autumn and winter 2002–2003, severe rainfall and flooding in to lower humid ombrotype). Associated endemic taxa are Cym- the area caused the destruction of the population in the lower balaria aequitriloba (Viv.) Cheval., Scrophularia trifoliata L., part of the channels, while the upper part was less damaged. Hypericum hircinum L., Teucrium marum L., Stachys corsica In 2004, however, the species re-appeared in the lower part, Pers., Digitalis purpurea L. var. gyspergerae (Rouy) Fiori, with numerous seedlings established in sand pockets and other Rumex scutatus L. ssp. glaucescens (Guss.) Brullo, Scelsi & favourable micro-sites. The Mt. Arcosu population shows nar- Spampinato. rower fluctuations thanks to the greater stability of the local rock formation. Demographic condition, conservation status and IUCN Anchusa capellii Moris, Stirp. Sard. El. 2: 6. 1827. — Type: category lecto-TO Herb. Moris. The Monte Lattias and Monte Arcosu populations consist of c. 1500 and 650 plants, respectively; their total surface of occu- Perennial. Hispid-pubescent for tubercle-based, hyaline setae pancy is c. 2.7 hectares (Table 2). The species is not currently up to 2 mm long, mixed to dense, shorter hairs. Stems up to included in any type of national Red List, but its rarity and 40 cm, prostrate-ascending to decumbent. Basal leaves form- small population size suggest that it should be included in the ing a rosette, oblanceolate, 7–14 × 1.5–3.5 cm, the cauline Systematics, phylogenetic relationships and conservation of the taxa of Anchusa (Boraginaceae) endemic to Sardinia (Italy) 165

Taxa n.s. n. p. n. ind. Area Trend Main factors of disturb and threat A. capellii 2 1 1.000 34.800 stable grazing, natural events A crispa ssp. crispa 9 3 2.100 60.000 declining habitat loss by human activity, alien species A. crispa ssp. maritima 5 5 6.000 90.000 declining habitat loss by human activity, alien species A. formosa 1 2 2.150 20.740 stable natural events A. littorea 6 1 350 2.800 declining habitat loss by human activities, alien species A. montelinasana — 1 200 43.950 ? grazing, natural events A. sardoa 1 1 1.500 30.000 declining habitat loss by human activities, tourism

Table 2 Total number of sites recorded previously (n.s.), number of populations currently conﬁrmed (n.p.), estimated number of fertile individuals (n.ind.), approximated area of occupancy of the populations (m2), demographic trend, and main factors of threat of the Sardinian endemics.

Taxa Habitat 92/43 National IUCN Proposed IUCN category ex situ A. capellii absent CR VU B2a; D1+2 CCB, FI, JBV A crispa ssp. crispa present EN CR B1ab(iv)c(iv)+2ab(iv)c(iv) FI, CCB A. crispa ssp. maritima present EN VU B1ab(iii)+2ab(iii); D2 FI, CCB A. formosa absent — VU B2ac(iv); D2 CCB, FI, JBV A. littorea absent CR CR B1ab(i−v)+2ab(i−v) CCB A. montelinasana absent — EN D CCB, FI A. sardoa absent — CR B2ab(iii) CCB, FI

Table 3 Presence/absence in the EU Directive Habitat 92/43, current IUCN category at the national level, category here proposed and location of ex situ collections of seeds or living plants of the Sardinian Anchusa endemics (CCB: Biodiversity Conservation Centre, Cagliari, Italy; FI: University Botanical Garden, Firenze, Italy; JBV: Jard´ıBotànic València, Spain). progressively smaller, subsessile. Cymes dense at first, but Chorology. Sardinian endemic. elongating and becoming lax in fruit. Bracts as long as or This species is known for only the top of Monte Santa Vittoria longer than calyx, ovate-lanceolate. Pedicels elongating in in central Sardinia (Nuoro province), where it was discovered fruit up to 8 mm, often deflexed. Calyx 6–7 mm divided to by Moris in 1825 (Figs 1,2). A second historical record by c. 2/3, broadly campanulate in fruit. Corolla tube 5–6 mm; Martelli dated 1888 from the near locality of Taccu de Sadali limb sky-blue, c. 12 mm diameter, subrotate. Anthers 2.2 mm, could not be confirmed despite our researches in that area partially overlapping scales. Heterostylous; stigma capitate- and in other sites with similar geomorphological features (e.g. bilobed. Mericarpids c.2.8× 1.8 mm, obliquely erect, with Monte Lusei and Monte Perdedu). a distinct basal annulus, surface sparsely tuberculate, dark- Habitat and ecology brown. Flowering occurs during April–June; fruiting occurs Anchusa capellii grows between 900 and 1200 m s.l.m. in during June–July. W-facing rocky channels with a mean inclination of c.35◦, Iconography. Selvi (1998: Fig. 6); Fig. 4D–E (original). on a metamorphic siliceous substrate (gneiss and fillades) of Paleozoic (Devonian) origin. The bioclimate is supramediter- Reproductive biology ranean (upper subhumid ombrotype). Plants are established in Pollination is, at least in part, by means of insects, either rock fissures and earth pockets among stones, together with Hymenoptera (Apis mellifera, Tetralonia malvae), Diptera several other herbaceous endemics such as Stachys corsica, (Bombylius sp.) or Lepidoptera (Maniola nurag). Cultivated (pers.) Cymbalaria aequitriloba (Viv.) A. Cheval., Hypericum plants are able to produce progeny through inbreeding. Seed annulatum Moris, Armeria sardoa Spreng., Petrorhagia saxi- set is high in either wild or cultivated plants, and percentage fraga (L.) Link ssp. bicolor (Jord. & Fourr.) Gamisans. of germination is always elevated (c. 85%). Seed dispersal is mainly facilitated by ants. Unlike the report of Selvi (1998), Demographic condition, conservation status and IUCN A. capelli is heterostylous, with short-styled (thrum) and long- category styled (pin) forms in an approximately 3:1 ratio. A dominance The single known population consists of c. 1000 individuals of the thrum form was also found in A. undulata ssp. hybrida distributed over c. 3.5 hectares (Table 2, Fig. 2). The factors (Dulberger, 1970), but this was not confirmed by Selvi (1998) that determine the sudden disappearance of this species about on a broader sample of the same taxon. As in other Anchusa 100 m below the top of the channels are unknown. The site species (Phillip & Schou, 1981; Selvi & Bigazzi, 2003) het- is not currently included in any type of protected area and is erostyly of A. capellii is ‘imperfect’ as it is not correlated with subject to grazing by sheep and goats. This is the main factor the position of the anthers in the corolla tube, which is at the of disturbance, which causes the destruction of the aerial parts same height in the two forms. of several plants. During the last eight years, however, the 166 G. Bacchetta et al.

Figure 3 Anchusa montelinasana sp. nov. in its natural habitat. population did not show substantial demographic fluctuations often pale-violet at the margins. Anthers c. 1.9 mm, inserted at and is likely to be in a steady state since a long period of time. the top of tube and partially overlapping scales. Homostylous. A remarkable reproductive efficiency is likely to contribute Style c. 6 mm long; stigma subtruncate-bilobed, with crowded, to this stability. This suggests that it should be ranked in the lageniform (flask-like) papillae with a crenulate-digitate plate- category VU (Table 3) instead of CR as recently proposed like cap. Mericarpids transversely ovoid, c.2.1× 1.7 mm, (Scoppola & Spampinato, 2005). blackish, coat surface finely tuberculate and with a reticulation of blunt ridges, and thin basal annulus. Flowering occurs Anchusa montelinasana Angius, Pontecorvo & Selvi, sp. nov. during May–June; fruiting occurs during June–July. – Type: Italia, Sardegna, Gonnosfanadiga (Cagliari), Monte Linas sotto punta Sa Cabixettas, 1070 m, 39◦26.559 N – Iconography. Figs. 3, 4A–C (original). 8◦37.598 E, coll. R. Angius, C. Pontecorvo et G. Mandis 20 Note. Anchusa montelinasana is morphologically close to A. Mai 2005 (holo- CAG, iso- FI, BM). capellii, but differs from the latter in a combination of quantit- Anchusae capellii similis, a qua tamen differt floribus ative and qualitative characters of taxonomic value (Table 4). minoribus homostylis, calyce ad 1/3 partito, corollae leviter Adult plants are mostly of smaller size, with a more slender violacea nec non caeruleis, nuculis minoribus nigrescenti- habit and shorter stems. Calyx is smaller (6.5 vs. 7.6 mm) and bus, annulo basali tenui. most important, less deeply divided (1/3 vs. 2/3 in A. capellii; Perennial herb with fusiform taproot. Indumentum of di- Fig. 4B, D); degree of calyx incision is an important taxo- morphic type, with tubercle-based, hyaline setae up to 2 mm nomic character in Anchusa (Gus¸uleac, 1929; Chater, 1972; long, and dense, shorter hairs throughout the plant; stems 10– Selvi & Bigazzi, 1998, 2003). Unlike in A. capellii, sepals 35 cm, numerous, branched from base, prostrate-ascending. are thickened along the median vein and have more evident Basal leaves in a dense rosette, oblanceolate, 5–10 × 1–2 cm, hyaline margins. The corolla is also distinctly smaller, reach- with slightly undulate margins bearing stiff bristles, tapering ing c. 8 mm in diameter vs. 11–12 mm in A. capellii,andis into a short petiole; the cauline sessile and progressively smal- characterised by a distinct violet component, which is absent ler, often almost linear. Cymes initially dense, but elongat- in the latter (flowers are sky-blue). Finally, mericarpids are ing and becoming lax in fruit. Bracts about as long as calyx, smaller and have a blackish coat surface, while the reticulation ovate-lanceolate, obscurely cordate at base, hirsute. Flower of A. capellii is dark grey-brown; the basal ring is distinctly pedicels 1–2 mm at anthesis, up to 8 mm in fruit and often de- more thickened in A. capellii. The last important character is flexed. Calyx divided to c. 1/3 into five triangular-ovate, acute the lack of heterostyly in A. montelinasana, as revealed by the lobes, c. 6 mm long, urceolate-tubulose in flower, up to 9 mm in examination of 100 fertile plants in the field. fruit; corolla bright blue-violet, with cylindrical tube c.5mm Like all the other endemics treated here, A. montelinasana and subrotate limb c. 8.5 mm in diameter, with rounded lobes, is diploid with 2n = 16 (Fig. 5). In karyotype formula this Systematics, phylogenetic relationships and conservation of the taxa of Anchusa (Boraginaceae) endemic to Sardinia (Italy) 167

Figure 4 A–C: Anchusa montelinasana sp. nov. (A) habit, (B) ﬂower with bract, (C) mericarpid; D–E: A. capellii (D) ﬂower with bract, (E) mericarpid. Scale bars = 10 mm; B, D = 5 mm; C, E = 1 mm.

species is identical to A. formosa (Selvi et al., 1997), but differs are effective pollen vectors. In preliminary tests seeds have from A. capellii in the presence of six metacentric and eight shown an elevated germination capacity. submetacentric chromosomes rather than eight metacentrics and six submetacentrics (Table 5). Chorology Reproductive biology (still poorly known) It is endemic to the top of Monte Linas in southwest Sardinia Flowers are frequently visited by Hymenoptera (Apis sp., (Iglesiente), where it was ﬁrst discovered by C. P. and R. A. in Halictus sp.) and long-tongued Diptera (Bombylius sp.), which May 2005 (Figs 1,2). 168 G. Bacchetta et al.

Character A. capellii A. montelinasana A. formosa Mean length of setae (mm) 2.1 2.0 3.1 Cymes elongating, lax elongating, lax, short, dense Flowers in all directions in all directions mostly unidirectional Bracts ≥ calyx ± as long as calyx < calyx Mean length of the calyx (mm) 7.6 6.5 6 Degree of incision of calyx 2/3 1/3 1/4 Pedicels up to 8 mm, deflexed up to 8 mm, deflexed 2–3 mm, patent Calyx in fruit campanulate, not costate tubulose-campanulate, ventricose-urceolate, with weakly costate reflexed teeth, strongly costate Mean diam. of corolla (mm) 12.2 8.5 8 Colour of the corolla sky-blue blu-violet blue-violet Mean length of the anthers (mm) 2.1 1.9 1.7 Mean size of the nutlets (mm) 2.8 × 1.8 2.1 × 1.7 2 × 1.5 Mean weight of the nutlet (dry) 0.00403 0.00254 0.00221 Heterostyly present absent absent

Table 4 Main differential characters between A. capellii, A. montelinasana and A. formosa.

Linas at c. 1180 m a.s.l.; it suddenly disappears below 1000 m. The geological substrate consists of granites and metamorph- ites of Paleozoic origin. The bioclimate is supramediterranean (lower humid ombrotype). The local plant community is dom- inated by herbs and low shrubs, and includes other endemics such as Echium anchusoides Bacch., Brullo & Selvi, Genista salzmannii DC., Silene morisiana Beg.´ & Rav., Thymus cath- arinae Camarda, Viola corsica Nym. ssp. limbarae Merxm. & Lippert.

Demographic condition, conservation status and IUCN category Anchusa montelinasana exists with a single population which occupies a surface of c. 4.4 hectares. It is subdivided in three close subpopulations placed along the upper part of adjacent channels and includes a total of only c. 200 fertile plants (Table 2; Fig. 2). In spite of such reduced size, ﬂowering and fruiting are abundant and this suggests that the population has probably been in a more or less steady demographic state for a Figure 5 Metaphase chromosome plate of A. montelinasana,2n= long time. The site is largely free from human disturbance and = 16. Scale bar 6 μm. included in the Site of Communitarian Interest Linas-Marganai Habitat and ecology (ITB041111) and in a proposed regional park (Regional Law The species grows on the rocky ﬂanks and among stones 31/89). We propose for this taxon the EN category (Table 3). in the upper part of three channels (named Su Campu de Is Anchusa sardoa (Illario) Selvi & Bigazzi, Pl. Biosys. Sermentus) that originate on the northeastern side of Monte 132: 136. 1998. – Type: lecto- TO Herb. Moris.

Taxa Karyotype formula Reference A. capellii (Mt. Santa Vittoria) 2n = 2x = 16:2M+6m+6sm+2stSAT Bigazzi et al. 2000 A crispa ssp. crispa (Isola Rossa) 2n = 2x = 16:2M+6m+6sm+2stSAT Bigazzi et al. 2000 A. crispa ssp. maritima (Badesi) 2n = 2x = 16:8m+4sm+2st+2stSAT Selvi & Bigazzi 1998 A. formosa (Mt. Arcosu) 2n = 2x = 16:2M+4m+8sm+2stSAT original A. littorea (S’Ena Arrubia) 2n = 2x = 16:6m+8sm+2stSAT Valsecchi, 1976 A. montelinasana (Mt. Linas) 2n = 2x = 16:2M+4m+8sm+2stSAT original A. sardoa (Porto Conte) 2n = 2x = 16:6m+6sm+2st+2stSAT Selvi & Bigazzi 1998

Table 5 Karyotype formula according to Levan et al. (1964) of the taxa of Anchusa endemic to Sardinia. Systematics, phylogenetic relationships and conservation of the taxa of Anchusa (Boraginaceae) endemic to Sardinia (Italy) 169

Figure 6 Anchusa littorea in its natural habitat.

Perennial. Hispid for dense, stiff, tubercle-based bristles Demographic condition, conservation status and and shorter hairs. Stems up to 30 cm, erect-ascending. Basal IUCN category leaves in a loose rosette, 10–15 × 1–2 cm, ovate-lanceolate, The only known population of A. sardoa consists of five sub- obtuse, with erose-dentate or crispate-undulate margins; cau- populations of c. 250 individuals each, distributed over a total line leaves similar but smaller, sessile. Cymes several, dense surface of c. 3 hectares (Table 2). Unlike A. capellii and at anthesis and scarcely elongating in fruit. Bracts foliaceous A. formosa, this species is in a declining demographic status, always much longer than calyx. Flowers subsessile. Calyx di- due to the heavy anthropic disturbance of the site where it vided to c. 1/2 into five subacute lobes, 9–11 mm long in fruit, grows. Tourism, car parking, pollution, afforestation, tramp- tubulose. Corolla pale blue with tube 4–5 mm and limb 5–7 ling and cleaning of the beach with mechanical means during mm diameter, subrotate. Anthers 1.7 mm, slightly overlapping the growing season are the main factors that are causing hab- scales. Style 4–5 mm long; stigma broadly ovoid with spaced itat loss and reduction of the population. This justifies the papillae. Mericarps obliquely ovoid, c.2.5× 1.5 mm, with application of the CR category (Table 3). a pointed apex, surface light brown-greyish, densely tuber- Anchusa littorea Moris, Atti Congr. Sci. Ital. Genova, 8: 566. culate, with a sparse reticulation of blunt ridges. Flowering 1846. – Type: lecto- TO Herb. Moris. occurs during May–June; fruiting occurs during June–July. Annual. Hispid-setose for dense, tubercle-based trichomes and Iconography. Selvi and Bigazzi (1998: Fig. 17). shorter hairs. Stems decumbent to suberect, branched from Chorology. Sardinian endemic (Nurra region). base, 4–15 cm. Lower leaves 3–6 × 0.4–0.8 cm, narrowly This species is known from only the coastal strip of the Porto oblanceolate and tapering into a short petiole, with repand- Conte bay in NW Sardinia (Fig. 1). dentate margins; cauline leaves almost linear, smaller and sessile. Cymes with small flowers distanced at the axyl of cauline leaves, often also in the lower part of the stems just above Habitat and ecology ground-level, on pedicels 2–3 mm long deflexed in fruit. Calyx Stable and flattened sand dunes are c. 100 m from the seashore, tubulose, 3.5 mm, divided to 1/2–2/3 into narrowly triangular mainly under artificial Pinus sp. pl. canopy. The bioclimate lobes, up to 5 mm and spherical-urceolate in fruit. Corolla with is thermomediterranean (upper dry-lower subhumid ombro- tube 4 mm long and limb 4–5 mm diam., light blue or white, type). rotate with rounded lobes. Anthers 1.3 mm, not overlapping scales. Style slightly longer than calyx. Mericarps light grey- brown, small, 1.5–2 × 0.5–1 mm, with a lateral beak and a thin Reproductive biology basal annulus, with finely tuberculate surface. Flowering oc- Flowers are abundantly visited by Hymenoptera and long- curs during March–May; fruiting occurs during March–June. tongued Diptera, which are likely to be the main pollen vectors. Like the other taxa, however, isolated plants in cultivation are Iconography. Moris (1837–59, Fig. 49); Valsecchi (1988, able to produce offspring through inbreeding. Fig. 1); Fig. 6. 170 G. Bacchetta et al.

Reproductive biology (almost unknown) in spring contemporarily to flowers, even through selfing, Although some insects’ activity has been observed in the single and then immediately released in the mobile sands of dunes known population, autonomous self-pollination is likely to exposed to seawind. They are easily moved with sand by wind be the main reproductive system. Seed dispersal is mainly and can remain latent for several years to germinate only when performed by wind and ants. edaphic humidity and position in terms of underground depth, distance from the sea and type of surrounding vegetation are in Chorology an optimal combination. This may account for the strong spa- Endemic to southwest Sardinia (Sulcis and Iglesiente), and re- tial and temporal discontinuity with which this species appears corded previously for the following localities: S’Ena Arrubia, along the windy coasts of southwestern Sardinia. Terralba, Marina di Arbus, Piscinas, Is Arenas, Sant’Antioco at Calasetta bay and San Pietro at Spalmatore (Fig. 1). Only Anchusa crispa Viv., App. Fl. Cors. Prodr. 1: 1. 1825. – Type: in one of these localities, however, is the species currently lecto- G-DC. confirmed (see below). Perennial to biennial. Hispid-setose for tubercle-based bristles, up to 2 mm long and dense, shorter hairs. Stems Habitat and ecology 10–40 cm, prostrate or prostrate-ascending. Basal leaves in Anchusa littorea is a psammophytic species growing on large, a loose rosette, 80–120 × 3–8 mm, sublinear or narrowly mobile dune systems of siliceous sand, in the discontinuities of oblanceolate, obtuse, with undulate-crispate margins; cauline Juniperus oxycedrus macrocarpa L. ssp. (Sibth. & Sm.) Neirl. leaves linear, sessile. Cymes several, dense or lax at anthesis, shrubs. The bioclimate is upper thermomediterranean (lower slightly elongating in fruit. Bracts triangular-ovate, subequal subhumid ombrotype). In the only locality where we could to flowering calyx. Pedicels up to 2 mm in fruit. Flower- Linaria flava oberve it, associated taxa are therophytes such as ing calyx 5–7 mm, up to 5 mm wide and 6–8 mm long in . sardoa , Phleum sar- (Poiret) Desf. ssp (Sommier) Arrigoni fruit, subglobose-urceolate, divided to 1/3–1/2 into five obtuse doum , Malcolmia ramosissima , (Hackel) Hackel (Desf.) Thell. lobes. Corolla blue or rarely whitish to violet, with tube 4–5 Tuberaria guttata , Polycarpopon alsinifolius (L.) Fourr. (Biv.) mm and limb 6–7 mm diam., rotate with subtruncate, rounded , Silene nummica DC. Vals. and others. lobes. Anthers c. 1.7 mm, slightly overlapping scales. Style 4–5 Demographic condition, conservation status and mm long; stigma capitate-ovoid. Mericarpids obliquely ovoid, IUCN category c.2.1× 1.3 mm, with a blunt apex, surface greyish, tuber- The rediscovery of A. littorea at Is Arenas comes after 8 years culated, with a reticulation of blunt ridges. Flowering occurs of unsuccessful field research at all the sites where it was recor- during April–July; fruiting occurs during June–July. ded previously and in other coastal localities of SW Sardinia. subsp. crispa. Basal leaves narrowly oblanceolate, 5–8 mm The last previous record was at ‘Spiaggia di Arborea’ near wide. Bracts ≥ flowering calyx. Calyx divided to 1/2–1/3, Oristano and dates back to April, 4, 1981 (L. Mossa, CAG!). with lanceolate lobes. Corolla limb c. 6 mm in diam. Stigma At that site, however, the species has never been observed usually placed at the same level or below the level of anthers. since. We failed to find it at most of the other localities (from Iconography. Valsecchi (1976: Fig. 5; 1988: Fig. 1); Selvi and north to south: Terralba, Marina di Arbus, Piscinas, San Pietro Bigazzi (1998: Fig. 15). and S’Antioco islands), and it has not been observed for over 25 years. The population found in spring 2005 consisted of only Reproductive biology c. 350 plants distributed over a surface of c. 2800 m2 (Table 2, The following insects were frequently observed to visit Fig. 2C). The area is legally closed to the public and included in the flowers: Bombus sp. (Hymenoptera, Apidae), two spe- the Site of Communitarian Interest ‘Piscinas-Rio Scivu’ (ITB cies of Osmia sp. (Hymenoptera, Megachilidae), two spe- 040071). In spite of this, the site is frequented by local people cies of Megachile sp. (Hymenoptera, Megachilidae)and for recreation purposes and is crossed by a footpath, which is Bombylius sp. (Diptera). However, the occurrence of the continuation of a service road. Anthropogenic disturbance autonomous self-pollination has recently been established contributes to the limitation of the habitat available for this (Quilichini et al., 2001). The species shows stylar polymorph- species, which is at the brink of extinction due to the extremely ism, which is partly correlated with geographic regions. In reduced range and small population size coupled with the lack ssp. crispa, the stigma is placed at the same level or below of ex situ collections. Its steep decline justifies the category the level of anthers (Quilichini et al., 2004). Seed dispersal is CR (Table 3) and underscores the need for specific actions of performed by ants (Quilichini & Debussche, 2000), rarely by conservation. water or cows, at least on Corsica (De Montmollin & Strahm, On the other hand, the remarkable rarity and fugacity of 2005). this species may also be associated with its peculiar biological Chorology. Cyrno-Sardinian endemic. and ecological features. In fact, this is the only member of the In Sardinia it is restricted to the northern coasts of the island group that shows a terophytic habit and a strongly abbreviated and recorded for the following localities (Fig. 1): Porticciolo, life cycle. A typical trait of its ‘ephemeral’ like biology is Porto Palmas, Stintino (Spiaggia della Pelosa) Tonnara Saline, the unique capacity to produce flowers and fruits shortly after Stagno di Pilo, Foce di Fiume Santo, Porto Torres, Isola Rossa seed germination, when plants are still in an apparently juven- and Torre Vignola. Our field research, however, allowed us to ile state (Fig. 6). The very small mericarpids are produced confirm only three of these localities (see below). Systematics, phylogenetic relationships and conservation of the taxa of Anchusa (Boraginaceae) endemic to Sardinia (Italy) 171

Number of ITS1 position

Taxa 21 63 77 88 114 160 183 201 274 GenBank A. capellii A T A T C A T A A AY383297 A. crispa ssp. crispa (Corsica) A G G C C A C G A AY071853 A. crispa ssp. crispa (Sardinia) – G G T C A C G C DQ882246 A. crispa ssp. maritima – G A C C C C G C DQ882247 A. formosa A G A T C A C A A AY383299 A. littorea – G A C C A C G C DQ882248 A. montelinasana – G A T C A C A C DQ882250 A. sardoa – G G C C A C G C DQ882249 A. undulata ssp. hybrida A G A C A A C G A AY383300

Table 6 Condensed alignment of variable positions (in column) in the ITS1 region of the in group endemics.

Habitat and ecology c. 7 mm in diam. Stigma usually placed above the level of Subspecies crispa is found on maritime sands and fairly firm anthers. dunes, and sandy fields near to the sea, often within the associ- Iconography. Valsecchi (1976: Fig. 9); Selvi and Bigazzi ation of Ammophiletum arundinaceae Br.-Bl. 1921 (Paradis & (1998: Fig. 15). Piazza, 1988). The bioclimate is thermomediterranean (upper Reproductive biology.Likessp.crispa. dry-lower subhumid ombrotype). Chorology. Sardinian endemic. Described as Anchusa maritima (Valsecchi, 1976), the Demographic condition, conservation status and taxon is closely related to ssp. crispa and intermediate forms IUCN category occur, especially at Isola Rossa and Torre Vignola. It grows The taxon is currently confirmed for only three localities: Isola in the large, sandy bay of the Coghinas River, Gallura. The Rossa, Porticciolo and Torre Vignola. At Isola Rossa the pop- following localities are confirmed: La Ciaccia, Valledoria, San ulation is established in two small sandy bays separated by a Pietro a Mare, Codaruina, mouth of the Coghinas River, Baia rocky promontory and consists of c. 1600 individuals, while delle Mimose, Badesi Mare at Li Junchi (Fig. 1). at Torre Vignola there are only c. 150 plants. In the small bay of Porticciolo, the population consists of c. 400 adult plants. Habitat and ecology.LikeA. crispa ssp. crispa. On the contrary, the species has apparently disappeared in the Demographic condition, conservation status and IUCN other localities where it was present previously: Porto Torres, category Foce di Fiume Santo, Stagno di Pilo, Tonnara Saline, Stintino This taxon occurs almost continuously in the Coghinas bay and Porto Palmas (Moris, 1837–59; Valsecchi, 1976). There from S. Pietro a Mare to Badesi. Here it forms a single large is little doubt that the destruction of the habitat caused by hu- population consisting of several subpopulations with a patchy man activities is the main reason for the extinction of these distribution. Like ssp. crispa it is facing increasing loss of hab- populations. A second negative factor is the competition of the itat and disturbance as a consequence of the anthropogenic use aggressive alien species, Carpobrotus acinaciformis (L.) L. of the sandy strip and invasion of Carpobrotus acinaciformis. Bolus, which is increasingly colonising the coastal habitats of However, we estimate a total number of individuals of c. 6000, Corsica and Sardinia (Bacchetta, 2001). On the contrary, there distributed along a coastal stretch of c. 9 km. The taxon is cur- is no evidence for the presence of the exotic ant, Linepitema rently listed in the ‘EN’ category (Scoppola & Spampinato, humile, in Sardinia, which has been observed to negatively af- 2005), but under IUCN criteria it should be placed in the VU fect the dynamics of seed dispersal of the Corsican populations category (Table 3). (Quilichini & Debussche, 2000). Finally, natural events may also cause the decline of populations of A. crispa, as in the case of the severe storms of December 1999 on the western coast of Phylogenetic relationships Corsica (Paradis & Piazza, 2000). The current category for this ITS1 sequences are deposited in GenBank-EMBL-DDB and taxon in Italy is EN (Conti et al., 1997; Scoppola & Spamp- can be retrieved using the accession numbers reported in inato, 2005), but the strong demographic decline and critical Table 2. The aligned sequence data set (available from the conservation status caused by the factors mentioned above jus- authors in ‘nexus’ format upon request) is 278 bp long. In the tify the category ‘CR’ in accordance with the recent evaluation Maximum Parsimony analysis, 217 positions are constant, 51 in De Montmollin and Strahm (2005). are parsimony non-informative and 10 are parsimony inform- subsp. maritima (Vals.) Selvi & Bigazzi, Pl. Biosys. 132: 136. ative. Thus, only a small proportion (3.6%) of the ITS1 region 1998. – Type: holo- SASSA, iso-FI has a useful phylogenetic signal in this group of Anchusa. Basal leaves linear, 3–4 mm wide. Bracts slightly shorter Variable positions within the ingroup, cut and condensed, are than calyx. Calyx divided to c. 1/3, with rounded lobes. Flowers showninTable6. 172 G. Bacchetta et al.

Figure 7 Neighbour Joining tree from ITS1 sequences. L = 75, CI = 0.91 and RI = 0.67. Bootstrap support values are reported above branches when > 50%.

The heuristic search produced 75 most parsimonious on the hypothesis of a common origin for this insular en- trees with Length = 75, Consistency Index (CI) = 0.91 and demic group were proposed on a morphological basis, although Retention Index (0.67). The Neighbour Joining tree is topo- no cladistic analyses were performed to test its monophyly logically identical to some of the most parsimonious phylo- (Selvi & Bigazzi, 1998). More recently, a phylogenetic study genies and is shown in Fig. 7; bootstrap values > 50% are of tribe Boragineae based on plastid and nuclear DNA markers shown above the branches. The monophyly of Anchusa subg. showed the weak divergence of the clade A. capellii / A. for- Anchusa includes the mainly continental A. undulata ssp. hy- mosa, but the incomplete taxonomic sample of that analysis brida, which receives a medium support (75% BS), as well as was a major obstacle to visualise relationships in the whole the clade of the insular species endemic to Sardinia, the object group (Hilger et al., 2004). The present reconstruction, which of this study (76% BS). The latter is split in two sister lineages, is the first one based on DNA sequences from the entire set of the first of which consists of the three mountain species, A. for- taxa known to date, suggests that the Sardinian endemics form mosa, A. capellii and the newly described A. montelinasana a monophyletic clade possibly sister to the euri-mediterranean (68% BS). Their ITS1 sequences differ in four positions. Pos. complex of A. undulata. Due to the low levels of variation of 21 is shared by A. formosa and A. capellii, while A. montelinas- ITS1 sequences recently observed in the whole group of An- ana is characterised by one bp deletion; pos. 63 and 183 are chusa sensu stricto (Hilger et al., 2004), the slight divergence shared by A. formosa and A. montelinasana, while it differs shown by the Corso-Sardinian clade acquires a relevant phylo- in A. capellii; pos. 274 is shared by A. formosa and A. capel- genetic meaning and finds support in the morphological syn- lii and differs in A. montelinasana. In the phylogenetic tree, apomorphies that discriminate the insular endemics from the this species is basal to the two others, which appear to be sis- rest of the genus. These are mainly represented by the special ters. The second lineage (BS < 50%) is formed by the littoral type of dimorphic indumentum, the prostrate-ascending habit, entities A. sardoa, A. crispa ssp. crispa, A. crispa ssp. mari- the brachymorphic flowers with relatively short tube and sub- tima and A. littorea, and is divided in two subgroups, which rotate, rather than hypocrateriform, limb, the small anthers, are not very consistent with either taxonomy or geographic and the tiny nutlets with a thin basal ring (Gus¸uleac, 1929; distribution. One includes A. littorea and the Sardinian acces- Valsecchi, 1976; Selvi & Bigazzi, 1998). A further ecological sion of A. crispa ssp. maritima from the Coghinas bay, while synapomorphy of the group, which is not found elsewhere in the second is formed by the eastern Corsican accession of the genus, is the edaphic preference for incoherent, siliceous A. crispa ssp. crispa, which is sister to a terminal clade with substrates, a factor which contributes to explain the absence the Sardinian accession of the same entity and A. sardoa. in the calcareous massifs of central-eastern Sardinia. From the karyological viewpoint, the finding of 2n = 16 in A. montelinasana supports that divergence and speciation in Sardinia Discussion has proceeded at the diploid level, as in most of the continental species of Anchusa (Smith, 1932; Britton, 1951; Luque, 1983; Systematics and phylogenetic hypotheses Selvi & Bigazzi, 2003). Coupled with the low levels of ITS1 The discovery of the new species A. montelinasana brings to divergence, lack of chromosome variation seems to indicate seven the number of taxa endemic to Sardinia, providing fur- that no abrupt genetic changes have occurred during processes ther evidence for the role of this island as a centre of diversity of morphological differentiation. On the basis of available of Anchusa in the Mediterranean. Preliminary considerations evidence, we assume therefore that the present-day endemics Systematics, phylogenetic relationships and conservation of the taxa of Anchusa (Boraginaceae) endemic to Sardinia (Italy) 173 originated in situ from a common ancestor through schizogen- volved in the effective adaptation of the populations to the etic speciation triggered by palaeogeographical events which local environment (‘outbreeding depression’, Barrett & Kohn, have caused the fragmentation and reduction of a formerly con- 1991; Frankel et al., 1995). Combined with high reproduct- tinuous range with consequent isolation of the populations. ive efficiency, this may explain the persistence and substantial Molecular data suggest that a main event in the evolution- demographic stability that we could observe in the mountain ary history of this group has been the split between the oro- endemics which have not experienced a severe deterioration phytic lineage of A. montelinasana, A. capellii and A. formosa, or loss of habitat for presumably a very long time. On the con- and the coastal, psammophytic lineage of A. crispa, A. littorea trary, the psammophytic taxa which are facing an increasing and A. sardoa. The question arose whether the former is basal anthropic pressure on the coastal ecosystems are in a more to the latter or vice-versa. Dune systems of western Sardinia precarious conservation status, despite their wider distribution started to form with the split-off of the Cyrno-Sardinian mi- in past times. The disappearance of several populations of A. croplate from southern France and northeast Spain in the late crispa and A. littorea in sites, which have been strongly altered Oligocene (c. 30 mya; Westphal et al., 1976; Cherchi & by human activities, shows that habitat maintenance is crucial Montadert, 1982; Speranza et al., 2002), suggesting that for the conservation of the populations still surviving along coastal species derived from orophytic progenitors occurring the north and western coasts of the island. Concrete actions on pre-existing massifs. Events of colonisation of the sandy such as fencing and limitation of invasive plants have been coastline may have triggered a process of speciation and ad- undertaken for A. crispa on western Corsica (Guyot & Mur- aptive radiation, which led to the origin of the psammophytic acciole, 1995; Paradis & Piazza, 2000) and these are urgent taxa. In view of the lack of endemics in the rugged mountains for also Sardinia especially in the case of the only known pop- of Corsica, despite their mainly granitic nature, and the much ulation of A. littorea. Coupled with the ex situ conservation lower diversification of the group on this island, our hypo- of the germplasm of all existing populations, these actions thesis implies that the Palaeozoic siliceous massifs of south should increase the chances of long-term conservation of this Sardinia, which were part of the Protoligurian mountain chain remarkable component of the Mediterranean island endemic connecting the western Alps to the eastern Pyrenees until the flora. late Oligocene (Westphal et al., 1976; Cherchi & Montadert, 1982) represent the centre of origin of the insular Anchusa clade. Acknowledgements This paper is dedicated to the memory of Professor Massimo Bigazzi, Conservation who prematurely died in April 2006. 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